Christelle M. Durand

Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders

SHANK3 (also known as ProSAP2) regulates the structural organization of dendritic spines and is a binding partner of neuroligins; genes encoding neuroligins are mutated in autism and Asperger syndrome. Here, we report that a mutation of a single copy of SHANK3 on chromosome 22q13 can result in language and/or social communication disorders. These mutations concern only a small number of individuals, but they shed light on one gene dosage–sensitive synaptic pathway that is involved in autism spectrum disorders.

Abnormal melatonin synthesis in autism spectrum disorders

Melatonin is produced in the dark by the pineal gland and is a key regulator of circadian and seasonal rhythms. A low melatonin level has been reported in individuals with autism spectrum disorders (ASD), but the underlying cause of this deficit was unknown. The ASMT gene, encoding the last enzyme of melatonin synthesis, is located on the pseudo-autosomal region 1 of the sex chromosomes, deleted in several individuals with ASD. In this study, we sequenced all ASMT exons and promoters in individuals with ASD (n=250) and compared the allelic frequencies with controls (n=255). Non-conservative variations of ASMT were identified, including a splicing mutation present in two families with ASD, but not in controls. Two polymorphisms located in the promoter (rs4446909 and rs5989681) were more frequent in ASD compared to controls (P=0.0006) and were associated with a dramatic decrease in ASMT transcripts in blood cell lines (P=2 × 10−10). Biochemical analyses performed on blood platelets and/or cultured cells revealed a highly significant decrease in ASMT activity (P=2 × 10−12) and melatonin level (P=3 × 10−11) in individuals with ASD. These results indicate that a low melatonin level, caused by a primary deficit in ASMT activity, is a risk factor for ASD. They also support ASMT as a susceptibility gene for ASD and highlight the crucial role of melatonin in human cognition and behavior.

Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments

SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability—more than 1 in 50—warrant its consideration for mutation screening in clinical practice.

SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism

Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies.

No human tryptophan hydroxylase-2 gene R441H mutation in a large cohort of psychiatric patients and control subjects

BACKGROUND It was recently reported that a rare functional variant, R441H, in the human tryptophan hydroxylase-2 gene (hTPH2) could represent an important risk factor for unipolar major depression (UP) since it was originally found in 10% of UP patients (vs. 1.4% in control subjects). METHODS We explored the occurrence of this variation in patients with affective disorders (n = 646), autism spectrum disorders (n = 224), and obsessive-compulsive disorder (OCD) (n = 201); in healthy volunteers with no psychiatric disorders (n = 246); and in an ethnic panel of control individuals from North Africa, Sub-Saharan Africa, India, China, and Sweden (n = 277). RESULTS Surprisingly, we did not observe the R441H variant in any of the individuals screened (3188 independent chromosomes). CONCLUSIONS Our results do not confirm the role of the R441H mutation of the hTPH2 gene in the susceptibility to UP. The absence of the variant from a large cohort of psychiatric patients and control subjects suggests that the findings reported in the original study could be due to a genotyping error or to stratification of the initial population reported. Additional data by other groups should contribute to the clarification of the discrepancy between our results and those previous published.

Expression and genetic variability of PCDH11Y, a gene specific to Homo sapiens and candidate for susceptibility to psychiatric disorders

Synaptogenesis, the formation of functional synapses, is a crucial step for the development of the central nervous system. Among the genes involved in this process are cell adhesion molecules, such as protocadherins and neuroligins, which are essential factors for the identification of the appropriate partner cell and the formation of synapses. In this work, we studied the expression and the genetic variability of two closely related members of the protocadherin family PCDH11X/Y, located on the X and the Y chromosome, respectively. PCDH11Y is one of the rare genes specific to the hominoid lineage, being absent in other primates. Expression analysis indicated that transcripts of the PCDH11X/Y genes are mainly detected in the cortex of the human brain. Mutation screening of 30 individuals with autism identified two PCDH11Y polymorphic amino acid changes, F885V and K980N. These variations are in complete association, appeared during human evolution approximately 40,000 years ago and represent informative polymorphisms to study Y chromosome variability in populations. We studied the frequency of these variants in males with autism spectrum disorders (n = 110), attention deficit hyperactivity disorder (ADHD; n = 61), bipolar disorder (n = 61), obsessive‐compulsive disorder (n = 51), or schizophrenia (n = 61) and observed no significant differences when compared to ethnically‐matched control populations. These findings do not support the role of PCDH11Y, or more generally of a frequent specific Y chromosome, in the susceptibility to these neuropsychiatric disorders.

Support for the association between the rare functional variant I425V of the serotonin transporter gene and susceptibility to obsessive compulsive disorder.

SIR—Recently, a rare functional variant, I425V, in the serotonin transporter gene (SLC6A4) has been reported to be associated with a complex neuropsychiatric phenotype that includes obsessive-compulsive disorder (OCD), alcohol abuse/dependence, anorexia nervosa, and pervasive developmental disorder (PDD).1 Our study, performed in a large population of patients with these disorders, confirms the occurrence and the segregation of V425 in OCD. The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has been implicated in numerous psychiatric disorders, mostly because of the efficacy of serotonin reuptake inhibitors (SRIs). Among the polymorphisms identified in SLC6A4, three have been shown to be functional. The rare I425V variation, located in exon 9, increases the transport activity of the protein2, whereas the two more frequent polymorphisms, 5-HTTLPR located in the 5′-UTR of the gene, and STin2 located in intron 2, modify the transcriptional activity of the gene3,4. Despite a large number of studies, the functional implications of the two frequent SLC6A4 polymorphisms in psychiatric disorders are still a matter of debate. In the present study, we sought to further explore the influence of the rare but clearly functional I425V variant in a large sample of patients with OCD and other psychiatric conditions previously reported in the two families carrying I425V, i.e., FDD, anorexia nervosa, and alcohol abuse/dependence. To be included in the study, patients had to meet the DSM-IV criteria for OCD, anorexia nervosa, alcohol abuse/dependence, or FDD. The diagnosis of FDD was confirmed using the Autism Diagnostic Interview-Revised.5 For the other disorders, lifetime psychiatric evaluation was carried out using the Diagnostic Interview for Genetic Studies (DIGS)6 for adult patients, or the Kiddie Schedule for Affective Disorders and Schizophrenia for children.7 Healthy controls were included after being interviewed with the DIGS and the Family Interview for Genetic Studies8 to confirm the absence of both personal and family history of major psychiatric disorders. The local Research Ethics Boards reviewed and approved the study. The V425 was found in 3/254 probands with OCD, 1/284 with PDD, 1/124 with anorexia nervosa, in 1/285 healthy controls, but not in alcohol abusers/dependents (0/128) (Table 1). In OCD family 1, V425 was transmitted by the father who also had a lifetime history of OCD and single phobia. The paternal grandfather was alcoholic and tobacco dependant. No genotypic information was available for the paternal grandparents since the grandfather died of throat cancer and the grandmother of breast cancer. In OCD family 2, the mother and two siblings of the proband had committed suicide and the father was also dead, so no genotypic information was available for these individuals. OCD case 1 had only one brother, who committed suicide some years ago, so no genotypic information was available for him. No other clinical data were available concerning the first-degree relatives of OCD case 1. In FDD family 1, V425 was transmitted by the father and was present in the proband and two brothers. The father and one of the brothers carrying the V425 were both alcohol dependent. However, the youngest brother (19 years old), also carrying the V425 variant, did not suffer from alcoholism or any other psychiatric disorder at the time of evaluation. Table 1 SCL6A4 genotypes and clinical features of the subjects included in the study Our results are in accordance with those previously reported by Ozaki et al1 on four points. First, we report a possibly higher occurrence of the V425 variant in OCD compared to controls. Although the variant is rare, the combined results of the two studies indicate a significantly higher frequency of V425 in OCD compared to controls1,9 (5/457 vs. 2/884, Fisher exact test, P= 0.02). Second, despite the limited clinical and genotypic information on the families carrying the V425 variant, our results suggest a possible co-segregation between the V425 and neuropsychiatric phenotypes, specifically in OCD. Third, Ozaki et al. hypothesized that the V425 variant may confer treatment-resistance to SRIs. This was indirectly supported by our findings since all OCD probands carrying the V425 variant in our study were considered resistant to SRIs, i.e., the severity of their obsessive and compulsive symptoms decreased less than 25% with multiple trials of a high dose of SRIs and a good compliance. Specifically, the three probands with OCD carrying the variant showed poor or no response to multiple trials of SRIs at adequate doses over several years. Fourth, the analysis of the two additional polymorphisms of the SCL6A4 gene indicated that the V425 polymorphism might be associated with the L allele of 5-HTTLPR. However, our results do not confirm the hypothesis of a combined gain of function effect of both V425 and L/L genotype as a genetic risk for OCD, since this combined genotype was not present in all affected patients and, by contrast, was present in the control subject carrying the V425 variant. In conclusion, our results are similar to those of the original report by Ozaki et al. and, therefore, lend support for a role of SLC6A4 V425 in the susceptibility to complex neuropsychiatric phenotypes. However, due to its global low frequency and to the fact that it was detected in a few controls, the role of V425 remains uncertain and should be interpreted with caution. We encourage other investigators, especially in the field of OCD, to screen for V425 in their samples. Indeed, the replication of these findings could ultimately implicate SLC6A4 as a true susceptibility gene to complex neuropsychiatric disorders, and consequently shed further light on the results obtained with the more frequent polymorphisms.

Genetic variations of the melatonin pathway in patients with attention-deficit and hyperactivity disorders.

Abstract:  Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration in melatonin signaling has been reported in a broad range of diseases, but little is known about the genetic variability of this pathway in humans. Here, we sequenced all the genes of the melatonin pathway –AA‐NAT, ASMT, MTNR1A, MTNR1B and GPR50 – in 321 individuals from Sweden including 101 patients with attention‐deficit/hyperactivity disorder (ADHD) and 220 controls from the general population. We could find several damaging mutations in patients with ADHD, but no significant enrichment compared with the general population. Among these variations, we found a splice site mutation in ASMT (IVS5+2T>C) and one stop mutation in MTNR1A (Y170X) – detected exclusively in patients with ADHD – for which biochemical analyses indicated that they abolish the activity of ASMT and MTNR1A. These genetic and functional results represent the first comprehensive ascertainment of melatonin signaling deficiency in ADHD.

Analysis of X chromosome inactivation in autism spectrum disorders

Autism spectrum disorders (ASD) are complex genetic disorders more frequently observed in males. Skewed X chromosome inactivation (XCI) is observed in heterozygous females carrying gene mutations involved in several X‐linked syndromes. In this study, we aimed to estimate the role of X‐linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 163 affected girls. The XCI pattern was also determined in two control groups (144 adult females and 40 young females) with a similar age distribution to the mothers sample and affected girls sample, respectively. We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X‐linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X‐linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (≥80:20) and a modest increased allele sharing was obtained in the Xq27‐Xq28 region, with a peak Z‐score of 1.75 close to rs719489. In summary, our results suggest that there is no major X‐linked gene subject to XCI and expressed in blood cells conferring susceptibility to ASD. However, the possibility that rare mutations in X‐linked genes could contribute to ASD cannot be excluded. We propose that the XCI profile could be a useful criteria to prioritize families for mutation screening of X‐linked candidate genes.

An investigation of ribosomal protein L10 gene in autism spectrum disorders

BackgroundAutism spectrum disorders (ASD) are severe neurodevelopmental disorders with the male:female ratio of 4:1, implying the contribution of X chromosome genetic factors to the susceptibility of ASD. The ribosomal protein L10 (RPL10) gene, located on chromosome Xq28, codes for a key protein in assembling large ribosomal subunit and protein synthesis. Two non-synonymous mutations of RPL10, L206M and H213Q, were identified in four boys with ASD. Moreover, functional studies of mutant RPL10 in yeast exhibited aberrant ribosomal profiles. These results provided a novel aspect of disease mechanisms for autism – aberrant processes of ribosome biosynthesis and translation. To confirm these initial findings, we re-sequenced RPL10 exons and quantified mRNA transcript level of RPL10 in our samples.Methods141 individuals with ASD were recruited in this study. All RPL10 exons and flanking junctions were sequenced. Furthermore, mRNA transcript level of RPL10 was quantified in B lymphoblastoid cell lines (BLCL) of 48 patients and 27 controls using the method of SYBR Green quantitative PCR. Two sets of primer pairs were used to quantify the mRNA expression level of RPL10: RPL10-A and RPL10-B.ResultsNo non-synonymous mutations were detected in our cohort. Male controls showed similar transcript level of RPL10 compared with female controls (RPL10-A, U = 81, P = 0.7; RPL10-B, U = 61.5, P = 0.2). We did not observe any significant difference in RPL10 transcript levels between cases and controls (RPL10-A, U = 531, P = 0.2; RPL10-B, U = 607.5, P = 0.7).ConclusionOur results suggest that RPL10 has no major effect on the susceptibility to ASD.